The prion protein (PrP) is a widely expressed and highly conserved cell surface glycoprotein of uncertain function. Aberrant metabolism of PrP is responsible for a variety of neurodegenerative diseases in both people and animals. These diseases include the transmissible 'prion diseases' such as bovine spongiform encephalopathy, as well as inheritable neurodegenerative diseases caused by mutations in the PrP gene. In neither case is the pathway(s) leading to cell death and neuronal damage understood. The overall goal of this project is to define the pathways of PrP-mediated neurodegeneration. To achieve this goal, we are studying the molecular pathways of PrP biosynthesis, intracellular trafficking, metabolism, and degradation. A quantitative analyses of these events are expected to shed light on how the various inherited mutations in PrP influence its biosynthesis or metabolism in a manner that leads the cellular dysfunction. [unreadable] [unreadable] Our analysis suggests that at least two cytotoxic forms of PrP (termed CtmPrP and cyPrP) are during the initial translocation of PrP into the endoplasmic reticulum (ER). Transgenic mice have now been created to determine whether CtmPrP-mediated neurodegeneration and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. We are also investigating the pathways by which the various forms of PrP are normally metabolized by the cell to determine whether modulation of these events are involved in the progression of neurodegeneration. It is anticipated that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to eventually cause neuronal dysfunction and death. Conversely, manipulation of these events may be able to slow or reverse the neurodegenerative process in these diseases.[unreadable] [unreadable] In parallel, we are also performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. The aim of these studies is to identify precisely the cellular locale and mechanism of PrP misfolding that initiates the disease process. Our current analyses have found that for a large number of mutants, misfolded PrP is found in a post-ER location, from where it is routed to lysosomes for degradation. This observation is notable because it suggests that the misfolded PrP species have somehow escaped the normal cellular quality control mechanisms in the ER, and instead use yet unidentified quality control pathways in the Golgi. These new pathways of quality control are now being investigated.[unreadable] [unreadable] In parallel studies, the downstream consequences of PrP misfolding and aggregation are being studied to identify the mechanism by which these events lead to cellular dysfunction. We have now found that these aggregates recruit various cellular factors, therby depleting their functional availability. One such factor is of particular importance because its disruption in mice leads directly to a neurodegenerative phenotype reminiscent of diseases caused by PrP.[unreadable] [unreadable] And finally, we are investigating the general properties of protein aggregates, which are associated with a wide range of diseases. We have discovered the the presence of cytosolic aggregates can significantly influence the pathways of normal cellular quality control. In one specific example, we have found that aggregates influence the fate of mislocalized secretory and membrane proteins. Rather than being rapidly degraded, the presence of aggregates causes these proteins to remain undegraded in the cytosol. This leads to their co-aggregation, facilitating the propogation of the existing aggregates and causing cell death. The molecular basis of this phenomenon is now being investigated.